The secreted Hedgehog (Hh) signaling molecules are essential to the coordination of cell-fate decision-making in multicellular organisms. Approximately 50% and 40% of medulloblastomas found in infants and children, respectively, are caused by deviant activation of the seven transmembrane Hh pathway effector Smoothened (Smo), the target of the clinically approved anti-cancer agent Vismodegib. An additional 40% of infant medulloblastomas harbor mutations in Suppressor of fused (Sufu), a protein that directly suppresses the activity of the Gli DNA binding proteins which are essential to Hh-dependent transcriptional activities. Infants with medulloblastomas are thus additionally challenged by the frequent presence of Sufu mutations which cannot be countered by Smo antagonists. Despite the rapid onset of tumor regression observed in children and adult patients treated with Vismodegib, the re-engagement of Gli transcriptional activity by Smo-independent mechanisms in the majority of treated medulloblastoma patients suggests drug resistance will be nearly universal. To delineate novel chemical space for managing Hh-related cancers that are refractory to Smo antagonists, we have completed a genome-scale cDNA library screen and have identified three out of the five anti-apoptotic Bcl-2 family members (Mcl-1, Bcl-2, and Bcl- xL) to drive Gli protein activation by directly engaging a previously unidentified BH3 sequence in Sufu. This interaction results in decreased Sufu ability to bind to and suppress Gli-mediated transcriptional activity likely by stabilizing a low Gli affinity conformation in Sufu. The transcription of the same Bcl-2 genes is regulated by Gli activity, thus revealing a concerted role of prosurvival Bcl-2 proteins in self-regulation through feed-forward suppression of Sufu activity. Small molecules targeting prosurvival Bcl-2 proteins (BH3 mimetics) disrupt this cancer-promoting signaling mechanism by breaking Mcl-1/Bcl-2/Bcl-xL interactions with Sufu and thereby enabling the means to inhibit Gli activity regardless of cancer cell sensitivity to Vismodegib. The studies outlined in this proposal will leverage novel BH3 mimetics as well as those in late stage clinical testing for evaluating the therapeutic promise of our findings using phenotypic and molecular read-outs in rhabdomyosarcoma and medulloblastomas, two Hh- associated malignancies.